Characterizing the DNA binding sites of activationinduced cytidine deaminase: A mutagenesis approach

Lucas, Heather M. (2013) Characterizing the DNA binding sites of activationinduced cytidine deaminase: A mutagenesis approach. Masters thesis, Memorial University of Newfoundland.

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Abstract

Activation induced cytidine deaminase (AID) is a 198 amino acid DNA mutating enzyme that converts deoxycytidine (dC) to deoxyuridine (dU) in singlestranded DNA (ssDNA). AID activity is important for adaptive immune responses with a key role during the affinity maturation of antibodies in activated Blymphocytes. AID targets the trinucleotide motifWRC (W=A/T, R=A/G) on (ssDNA), but neither its enzymatic mechanism(s) nor its modes of binding to ssDNA are known, largely due to a lack of protein structural information. A model of the enzymatic structure of AID suggested two potential ssDNA binding grooves on its surface. To confirm or refute the presence of two substrate binding grooves (Groove 1 and 2); amino acid residues were mutated singly or in combination in Grooves 1 and 2 to evaluate changes in DNA binding affinities and AID enzyme activity velocity compared to wild-type (wt) protein. Some AID mutants were inactive and had little or no binding to DNA, while others retained these properties relative to the wt enzyme. Contrary to our hypothesis, electrostatic interaction is not integral to AID and substrate interaction as previously believed. Along with activity and binding, conformational changes were confirmed in AID mutants, which could explain the differences in enzymatic functionality. Our research suggests AID conformation is key for substrate targeting and binding, and that putative Groove 1 likely contains contact points with ssDNA substrate.

Item Type: Thesis (Masters)
URI: http://research.library.mun.ca/id/eprint/11765
Item ID: 11765
Additional Information: Includes bibliographical references (pages 100-109) -- Restricted until October 2015.
Department(s): Medicine, Faculty of
Date: July 2013
Date Type: Submission
Library of Congress Subject Heading: Mutagens; Mutagenicity testing; Binding sites (Biochemistry); Enzymes

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